An antibody (immunoglobulin) is a protein specifically binding to an antigen and plays the leading role in humoral immunity in-vivo. FIG. 6(a) is a schematic view showing a basic structure of an antibody (immunoglobulin). An antibody molecule 61 has two heavy chains (H-chain) 62 having a molecular weight of 50,000 to 70,000 and two light chains (L-chain) 63 having a molecular weight of 23,000. These four chain-form polypeptides are connected via a disulphide bond and a non-covalent bond to form the shape of the letter Y as a whole. The heavy chain (H-chain) 62 is constituted of 4 or 5 domains. A variable region (VH) domain is positioned on the N-terminal side and 3 to 4 constant-region (CH1, CH2, CH3, CH4) domains are arranged toward the C-terminal. Note that, FIG. 6(a) shows a heavy chain 62 constituted of four domains (VH, CH1, CH2, CH3). The light chain (L-chain) 63 is constituted of two domains. A variable region (VL) domain is positioned on the N-terminal side, and a constant region (CL) domain is positioned on the C-terminal side.
The variable regions (VH and VL) of the heavy chain 62 and the light chain 63 are positioned at two sites on the N-terminal side and integrated with each other to sterically form an antigen-binding site specifically binding to an antigen. Therefore, specificity of the antibody is determined by the amino acid sequences of the heavy-chain variable region and the light-chain variable region (VH and VL) and a combination thereof. The amino acid sequences of the variable regions (VH and VL) and the combination thereof vary depending upon the corresponding antigen. The constant regions (CH1, CH2, CH3, CH4 and CL) of the heavy chain and the light chain have almost the same structure in every class or subclass.
The antibody molecule 61 is decomposed by a protease. i.e., papain at the hinge 64 present between CH1 and CH2 domains of the heavy chain to obtain two Fab fragments 65 and a single Fc fragment 66, as shown in FIG. 6(b). Note that, when the antibody molecule 61 is decomposed by another type of protease, i.e., pepsin, an F(ab′)2 fragment 67 can be obtained, which has two Fab fragments 65 connected via a disulphide bond at the hinge 64, as shown in FIG. 6(c). The Fab fragment 65 and the F(ab′)2 fragment 67 have an antigen-binding site constituted of the heavy-chain and light-chain variable regions (VH and VL) and thus have a specificity to an antigen. They can be used in an antigen-antibody reaction.
Furthermore, a single-chain antibody (scFv) 68, which has a heavy-chain variable region (VH) and light-chain variable region (VL) connected via a linker peptide 69, can also form a sterical antigen-binding site constituted of the heavy-chain and light-chain variable regions (VH and VL) and thus has a specificity to an antigen. The antibody (scFv) 68 can be used for an antigen-antibody reaction. FIG. 6(d) is a schematic view showing a basic structure of a single-chain antibody (scFv) 68. In FIG. 6(d), the C terminal of the heavy-chain variable region (VH) is linked to the N terminal of the light-chain variable region (VL) by a chain-form linker peptide 69.
In the antigen-antibody reaction, the specificity between an antigen and an antibody is high. If an antigen-binding site varies, the corresponding antigen differs. There are not less than a million combinations of a heavy-chain variable region and a light-chain variable region (VH and VL). Which type of antigen-antibody reaction is exhibited by each of these combinations must be verified by a basic experiment. Furthermore, taking advantage of high antigen-antibody specificity, the antigen-antibody reaction has been used in various applications and fields.
As a method for detecting or measuring a minute amount of substance, immunoassay is conventionally known, which uses specific affinity between an antigen and an antibody. The immunoassay uses the diversity of antigen-antibody reaction to analyze various biogenic substances and is used in a wide variety of fields. Furthermore, to increase measurement sensitivity of immunoassay, various types of methods using a label such as a radioactive compound, a fluorescent substance, an enzyme are known. Corresponding to the labels, they are called e.g., radio immunoassay (RIA), immunofluorescent assay (fluoroimmunoassay: FIA) and enzymatic immunoassay (ELISA: Enzyme-Linked ImmunoSorbent Assay and also called enzyme immunoassay). Particularly, ELISA is a highly sensitive method excellent in quantitativity and a highly versatile detection method requiring no complicated steps such as purification and pretreatment and thus used in various analyses such as medical diagnosis, quantification of environmental hormones/residual agricultural chemical, bovine spongiform encephalopathy (BSE) examination and proteome analysis.
In these microanalyses based on immunoassay, a solid-phase method is employed, in which a protein such as an antigen, an antibody or an enzyme is immobilized to a solid phase such as a test tube or a microplate by means of physical adsorption and chemical binding. As the solid phase, usually, a hydrophobic plastic is used. Since a protein tightly binds to the hydrophobic solid phase by means of a hydrophobic interaction, the hydrophobic solid phase can be excellently used in a relatively large number of proteins. Presently, hydrophobic polystyrene (PS) is frequently used as a hydrophobic plastic.
Furthermore, any one of the immunoassays requires a plurality of times of an adsorption/reaction treatment and a washing treatment and thus takes a long time until a target substance is detected. Note that, also in an experiment for verifying an antigen-antibody reaction, the experiment is generally performed by immobilizing an antibody or the like to a solid phase such as a test tube and a microplate. In short, a solid-phase method is used.
For example, ELISA is roughly divided into a direct adsorption method, a sandwich method and a competitive method. In the case of the sandwich ELISA method according to Patent Document 1, first, an antibody against a target substance is allowed to bind to a solid phase and then the solid phase is washed a plurality of times. Second, a reagent (hereinafter referred to as a “blocking reagent”), which is not involved in an antigen-antibody reaction and an enzyme reaction, is allowed to bind to unbound sites remaining on the solid phase such that other reagents may not bind to the surface of the solid phase. After completion of blocking, the solid phase is washed a plurality of times. Third, a sample containing the target substance is allowed to react with the antibody bound onto the solid phase, and then, the solid phase is washed a plurality of times. Fourth, a second antibody is allowed to react with the target substance, and then, the solid phase is washed a plurality of times. Fifth, the second antibody is allowed to react with an enzyme label, and then, the solid phase is washed a plurality of times. Sixth, the enzyme label is allowed to react with a substrate and absorbance is measured to detect the target substance in the sample or the concentration thereof is measured (see Patent Document 1, paragraph 0039). Note that, if an antibody previously labeled with an enzyme is used as the second antibody to be reacted in the fourth treatment, the fifth treatment is no longer required.    Patent Document 1: National Publication of International Patent Application No. 2002-526777